About RNA extraction - Trizol (Aug/24/2008 )

Hi everyone,

I am doing RNA extraction using TRIzol solution. However, I faced some problems.

1. The TRIzol (Originally in pink colour) turns darker (bloody red) after adding to lyse my MCF-7 cell (treated with my target drug that is drak green in colour). I have washed the cell with DEPC-water twice before adding TRIzol. Do anyone face similar problem and would my target drug interact with TRIzol?

2. After adding isopropanol to precipitate my aquoues phase of RNA and centrifuging the isopropanol, a transparent and droplet-like pellet was found in the bottom but this pellet is not stick to the bottom and can easily to lose if directly discard the supernatant. In the past after centrifuge the isopropanol, a small and white pellet which stick to bottom tightly is seen and would not lose by direct discarding the supernatant . If the transparent and droplet-like pellet is a contamination?

Thanks a lot

Panda@.@

I have seen TRIZOL turn from pink to dark brown (dry blood) after mixed with samples and still working.
I will suggest do TRIZOL twice as protocol indicated, and see what color it turn at second time.

There are something will react with TRIZOL, but only the company can tell you for sure will your durg interact with TRIZOL.

For the pellet, a very very pure and clean RNA pellet is a transparent and droplet-like pellet, but still change to white and small stuff during ethanol washing.
Is your pellet behaves like that during ethanol wash?

As I understand, brand of tube or force of centrifuge (time and rpm together) could leave pellet not attach to tube. I am not sure it is normal or not, for my RNA pellet, it never attach to tube during ethanol wash.

I will see if there is any problem with your RNA first. I only come back to those problems/questions if RNA is not good or no RNA at all.

Life is short, focus on result not processing.

Hello,

I`m new here and I have a question to the RNA-Isolation with TRIzol and so I`m writing my question in this post, too.

I have done some RNA-Isolations from bivalvia and some were good, but in the last days something was wrong.

I`m working after the script from invitrogen and do my steps like they say.
Step 2 with the Phase separation isn`t so good. I should give Chloroform to my sample (tissue and trizol) and should shake them and then centrifuge this. After them the mixture should seperate in a lower red, phenol-chloroform phase, an interphase and a colourless upper aqueous phase ( with my RNA).

But after my centrifugation I have a colourless/ light pink phase, an interphase and a upper red phase. I repeat this step, but it wouldn`t be better.

We finished then the other steps but we haven`t anything on the gel.

Can you help me?

Thanks,
Heike